1. Technical Field
The present disclosure relates to polypeptides derived from OspC (“outer surface protein C”) from bacteria of the genus Borrelia, in particular a protein which comprises a first OspC polypeptide, wherein the first OspC polypeptide is linked to a second OspC polypeptide via a disulphide bridge. The disclosure also relates to a method for the detection of antibodies against OspC and a method for the detection of a Borrelia infection, wherein a protein according to the disclosure is employed, and also to a diagnostic kit and a vaccine against Borrelia. 
2. Description of the Related Art
Bacteria of the genus Borrelia are described as pathogens of several human diseases, in particular as pathogens of Lyme borreliosis and of relapsing fever. An infection nowadays is usually detected via determination of specific antibodies against the bacteria in human or animal body fluids. In this context, the presence of specific antibodies against antigens which occur only in Borrelia indicates an infection with Borrelia. Early phases of infection, in particular fresh infections up to four weeks after initial contact, are characterized by the presence of antibodies of the IgM class, in particular against the antigens OspC, p41 (flagellin) and P39 (BmpA), while late phases, in particular infections which have run their course, have been cured or are chronically manifest, are accompanied by the presence of antibodies of the IgG class, in particular against the antigens VIsE, p83/P100, p58, OspA, p41 (flagellin), P39, P18 and others (Wilske and Fingerle, 2005).
An early diagnosis of the disease is particularly important because therapy with antibiotics is more successful and easier in the early stages than in late stages, thus, e.g. in the early phase, an oral therapy with antibiotics is still possible.
Antibodies against OspC of the IgM class are generally the most important marker for an early phase of the disease. Native OspC is a membrane protein of the lipoprotein family 6 acylated with a fatty acid, which is anchored in the outer cell membrane (Norris et al., 1992; Hagman et al., 1998). During expression, in parallel with the cleavage of the signal sequence by signal peptidase II, the acyl residue is attached to the only conserved cysteine occurring in the OspC amino acid sequence (Wu and Tokunaga, 1986). It serves for anchoring in the lipid-containing cell membrane.
A conserved C-terminal peptide of ten amino acid residues, the terminal carboxyl group of which must be freely accessible, is generally regarded as the most important epitope within OspC for reactions with human antibodies of the IgM class (Mathiesen et al., 1998a; Mathiesen et al., 1998b), as is also described in the PCT patent specification WO 9742221. Epitopes in the poorly conserved central region of OspC are also described (Earnhart et al., 2005).
For detection of the antibodies, antigens purified from Borrelia are regularly used in immunobiochemical tests, such as ELISA, line blot or western blot. In the methods, individual or several antigens are conventionally bound to a solid phase and brought into contact with the body fluid to be analysed, and the bound antibodies are detected by a reporter molecule. Such kits are marketed e.g. by EUROIMMUN AG as EUROLINE-WB and Anti-Borrelia-plus-VIsE-ELISA. Similar methods are described in the literature (Hansen et al., 1988; Cutler and Wright, 1989; Fister et al., 1989).
Methods which are based on non-recombinant preparations can be reproduced only with a high outlay and are therefore cost-intensive, because essentially complex culture media which contain natural constituents which are not chemically defined, such as protein-containing serum fractions from Mammalia and complex protein mixture, e.g. proteolytically treated muscle extracts, yeast extracts or gelatine, are used for in vitro culturing of Borrelia. A commercial kit on this basis is obtainable e.g. from Sigma Aldrich (Complete BSK-H). Similar compositions are described in the literature with the designations MKP and BSK II (Ruzic-Sabljic and Strle, 2004).
Unfortunately, the constituents of these media which are not chemically defined are subject to marked variations in their compositions from batch to batch, are at risk of contamination with viruses or Mycoplasma and are cost-intensive to produce. Accordingly, Borrelia cultured in vitro conventionally show marked variations in their growth rates and their gene expression patterns, depending on the particular culture media used or depending on individual components of the particular culture media used. Genes which are expressed in vivo, depending on the particular host organism, are particularly affected by this (Pollack et al., 1993; Yang et al., 2001). This applies in particular to OspC. The culturing temperature and the oxygen and carbon dioxide concentration furthermore also have a decisive influence on the nature and amount of the proteins expressed (Seshu et al., 2004; Hyde et al., 2007). As a result, in particular the results from different laboratories which culture Borrelia can be poorly compared with one another.
Methods which are based on individual antigens purified from Borrelia cultured in vitro are furthermore susceptible to non-specific reactions caused by the presence of further impurities which have not been adequately depleted. In particular, in methods which only generate one signal, such as, for example, ELISA or line blot, falsely positive results are thus regularly obtained. In contrast, western blots, which bypass this problem by local resolution of the signals, have the disadvantage that they generate a considerable additional effort due to the required electrophoresis and the transfer to a membrane.
In further methods, antigens are used which are prepared by recombinant techniques, e.g. by heterologous expression of antigens or antigen fragments in E. coli, as described e.g. in the European patent specification EP 0506868. Commercially obtainable variants are e.g. EUROLINE-WB and Anti-Borrelia-plus-VIsE-ELISA (IgG) and the recomline Borrelia IgM (Mikrogen). Similar methods are described in the literature (Hauser et al., 1998; Lawrenz et al., 1999; Wilske and Fingerle, 2005). In general, recombinant antigens have the advantage that they can be purified in a more defined manner and with less effort than native antigens, e.g. by fusion with purification polypeptides, e.g. the polyhistidine-tag (=His-tag).
Heterologous expression of OspC including its intrinsic signal sequence in E. coli and subsequent acylation is possible, but leads to a low expression efficiency (Fuchs et al., 1992). For this reason, in general deletion constructs of OspC with increased expression efficiency in which at least the signal sequence is missing are employed. In this context, in the prior art, at least the first 19 amino acids of OspC, including the cysteine which occurs in the OspC amino acid sequence, are deleted (Fuchs et al., 1992; Wilske et al., 1993; Wilske et al., 1995; Eicken et al., 2001; Kumaran et al., 2001), and this is in some cases supplemented by N-terminal fusion with heterologous polypeptides, e.g. the His-tag.
Such N-terminally shortened variants have also been used for clarification of the spatial structure of OspC (Kumaran et al., 2001; Eicken et al., 2001). For this, the first 31 (Eicken et al., 2001) or 37 (Kumaran et al., 2001) amino acid residues of OspC were deleted in the recombinant OspC variants in order to achieve in particular a suitable amount and purity of the OspC. The two exemplary spatial structures indicate that recombinant OspC tends to dimerize. This dimerization takes place on the basis of ionic interactions and hydrogen bridge bonds, but not by a covalent linking.
There are also attempts to prepare acylated variants of OspC recombinantly by upstream insertion of a heterologous signal sequence, as described e.g. in the European patent specification EP 1741718. By attaching the fatty acid, such OspC variants are intended to have a higher in vivo immunogenicity than the N-terminally deleted or modified OspC variants described until then.
The recombinant variants of OspC used hitherto for diagnostics in general have the disadvantage that they differ from the native OspC purified from Borrelia cultured in vitro, because they are not or are differently modified post-translationally, and the presence of conformational epitopes or the accessibility of epitopes in general is not comparable or not guaranteed. The manifestation of these disadvantages is the lower specific reactivity of recombinant OspC variants used to date compared with non-recombinant preparations, which is regularly described in the literature. It is reflected in high rates of false positive results in such diagnostic methods.
The development of recombinant OspC variants which, with an easier and more defined preparation compared with the native antigens, can be employed in diagnostic methods and at the same time do not lead to the disadvantages described for the recombinant OspC variants to date is therefore necessary in order to facilitate the preparation of such in vitro diagnostic agents or the implementation of such diagnostic methods and to standardize the results of different laboratories.